RING E3 ubiquitin ligases provide specificity to a huge fraction of biological ubiquitination reactions. Working in a manner that does not involve a covalent intermediate, these molecules bind substrates and specific E2 ubiquitin conjugating enzymes to effect transfer of ubiquitin to Lys residues on substrates, on the E3 (autoubiquitination), and on ubiquitin (polyubiquitination). The distribution, kinetics and specificity of these potentially competing reactions determine whether products will be targeted for proteolysis, differentially localized, and/or signal various forms of stress. We cloned Chfl and Chf2, the yeast homologs of the Chfr tumor suppressor, and developed genetic systems that indicate that Chf-imposed cell cycle delays depend on function of both Ubc4 and Mms2. We reconstituted purified ubiqutination reactions using Ubc4 and Ubc13/Mms2 as the ubiquitin conjugating enzymes and Chf1 and Chf2 as E3 and identified the reaction products by high resolution mass spectrometry. We have also mapped a specificity-determining region to the N-terminus of Chf1, identified large sets of Chf1 and Chf2 interactors, and developed novel methods to quantitate and provide a kinetic description of complex site-specific ubiquitination reactions. Specific Aims: 1) We will use quantitative mass spectrometry and enzymologyto define the sites, linkages and kinetics of Chf1 and Chf2 ubiquitination reactions with genetically validated ubiquitin conjugating enzymes and the proteins we have identified as Chf interactors. 2) We will determine the sites, linkages, biological consequences, and E2-dependence of Chf1 and Chf2 ubiquitination in vivo. This proposal has two long-term public health objectives. First, determining the mechanisms of function of yeast Chf1 and Chf2 is critical to understand function of Chfr, which is frequently inactivated in human tumors of epithelial origin. Second, innovations in analysis of RING E3 ubiquitin ligases are necessary to understand the specificity of function of RING E3 ubiquitin ligases, which have key functions in the health of every organ system in the human body.